Hemophilia (HA) is caused by loss-of-function mutations in the X-linked Factor (F) VIII gene, F8. Infusion of replacement plasma-derived (pd) or recombinant (r) FVIII is the standard of care to manage this chronic disease. Infusion of replacement FVIII, however, is not a cure for HA. Spontaneous bleeding remains a serious problem especially for those with severe HA, defined as circulating levels of FVIII coagulant activity (FVIII: C) below 1% of normal. Furthermore, the formation of anti-FVIII antibodies occurs in about 20% of all patients and more often in certain subpopulations of HA patients, such as African Americans (Viel K R, Ameri A, Abshire T C, et al. Inhibitors of factor VIII in black patients with hemophilia. N Engl J Med. 360: 1618-27, 2009). Patients unable to be treated with FVIII experience more painful, joint bleeding and over time, a greater loss of mobility than patients whose HA is able to be managed with FVIII.
The approval in Europe of the first gene therapy obtained by uniQuire BV for alipogenic tiparvovec (trade name Glybera) to treat lipoprotein lipase (LPL) deficiency is a milestone in the quest to bring gene-based therapeutics into clinical use. Tiparvovec (AAV1-LPL(S447X)) incorporates an intact human LPL gene (LPL) variant, i.e. LPL(Ser447X), in an adeno-associated virus (AAV) vector, which is delivered intramuscularly (Gaudet D, Méthot J, Déry S, et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1LPL(S447X)) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther. Jun. 21, 2012. [Epub ahead of print]). The benefits of restoring continuous circulation of clinically meaningful levels of FVIII has motivated intense efforts over decades to develop an effective gene therapy for hemophilia. Use of an AAV vector to deliver the gene that encodes FIX is bearing fruit in the treatment of hemophilia B (HB) (Nathwani A, Tuddenham E G D, Rangarjan S, et al. Adeno-associated viral vector mediated gene transfer for hemophilia B. Blood. 118(21): 4-5, 2011). HB in six adult patients has been converted from a severe form to mild or moderate HB, following intravenous infusion of an AAV8 vector incorporating human F9 under the control of a liver restricted promoter (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). These patients have maintained a circulating FIX coagulant activity (FIX: C) level ranging from 1-6% of normal for 3 years.
Although very encouraging, the AAV vector is not suitable for many HB patients and safety concerns remain. AAV vectors have been engineered from a wild-type parvovirus capable of naturally infecting humans (Calcedo R, Morizono H, Wang L, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol. 18(9): 1586-8, 2011; High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). In the current AAV trial, patients are screened for neutralizing antibodies against AAV. Thus, about 30-50% of hemophilia patients may not be eligible for this treatment (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). Patients with liver disease are also not eligible for this therapy pending a better understanding of safety. Furthermore, because the vectors are predominantly non-integrating, they are not suitable for use with young patients because expression of FIX would be expected to be lost as the patient grows (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012).
Transmission of AAV to the germ-line (semen) has been reported, but this appears to be transient (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). There is a trend toward a dose-dependent memory T-cell mediated response to AAV. This was seen in the first HB trial done with AAV and again in the current trial for HB (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). Whereas dosing in four patients in the range of 2×1011 vg/kg did not provoke a T-cell response, it provided only low circulating FIX: C levels (1-3%). Dosing at 2×1012 vg/kg in two patients led to initial robust levels of FIX in circulation, 8-10%; but, at 8-weeks post infusion, FIX levels began to fall and patients' liver enzymes rose (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). Treatment with prednisolone was effective in restoring normal liver enzyme levels and reducing AAV-capsid specific T-cells within PBMCs (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). In one patient, FIX fell to 2% of normal, but the other maintained FIX levels at 6% of normal (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012). Questions remain about the long-term safety of therapy with AAV. Sequencing studies of organs from animals treated with AAV demonstrate that integration of AAV occurs (Naki H, Yant S R, Storm T A, Fuess S, Meuse L, Kay M S. Extrachromosomal recombinant adenoassociated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol. 75(15): 6969-76, 2001). There is also one report of neonatal mice injected with AAV2 experiencing an increase in hepatocellular carcinoma with some tumors containing vector DNA (Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012). Whether AAV will prove to have any utility for delivering F8 to HA patients is an open question. The cloning capacity of the vector is limited to replacement of the virus' 4.8 kilobase genome. Promising preclinical results have been obtained using an AAV packaged, non-naturally occurring F8 cDNA, encoding B-domain-deleted (BDD)-rFVIII. One major caveat is that the dosing required to achieve desired levels of circulating FVIII (1-7.8%) in these studies was a log higher than the highest dose used in the current HB trial (Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012; Jonathan D. Finn, Margareth C. Eradication of neutralizing antibodies to FVIII in canine hemophilia A after liver gene therapy. Blood 116: 5842-5848 2010). Experience in the HB trial suggests that a memory T-cell response targeting liver cells would occur at these doses (High K A. The gene therapy journey for hemophilia: are we there yet? Blood. 120(23): 4482-7, 2012; Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012).
A substantial effort has been made to develop lentiviral vectors for the delivery of clotting factors (Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012). Lentiviral vectors can transfect non-cycling hepatocytes and integrate into the host genome. While the latter attribute may afford long term expression of the factor, it raises serious safety concerns regarding insertional mutagenesis leading to activation of oncogenes or inactivation of repressors. Studies in an HB mouse indicate that a preferred lentiviral vector could be one that harbors an inactivation mutation of the integrase, termed an IDLV (integrase defective lentiviral viral vector) (Matria J, Chuah M K, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther. 18: 477-90, 2010). Although factor expression is diminished following the elimination of integration, it appears this may be offset in the case of FIX by use of a rare hyper-activating variant in a codon-optimized F9 cDNA which dramatically boosts circulating FIX levels (Matria J, Chuah M K, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther. 18: 477-90, 2010). Another obstacle with lentivirus is its competence to transfect APCs which triggers deleterious T-cell mediated immune responses that can neutralize the secreted clotting factor; or eliminate the transduced hepatocytes (VandenDriessche T, Thorerezn L, Naldini L, et al. Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine track can efficiently transduce non-dividing hepatocytes and antigen presenting cells in vivo. Blood. 100: 813-22, 2002). This can be reduced in animal models by including an additional layer of post-transcriptional control, mediated by the use of endogenous microRNA (miR) (Matria J, Chuah M K, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther. 18: 477-90, 2010). Whether manipulations designed to mute the immune response to lentivirus will translate to man is unknown. In contrast to what occurs in man, in the canine model, AAV vectors did not elicit an immune response. Hence, while animal models have proven predictive with regard to forecasting delivery of factor clotting activity; no animal model, including the canine, recapitulates the more refined and sophisticated human immune response.
Use of autologous cells engineered with viral elements or nucleases capable of genomic editing may permit greater safety than intravenous delivery of targeted virus. Ex vivo protocols allow for screening of the genomes of manipulated cells to assess the frequency or viral insertions, double strand breaks in DNA (DSBs) or other potentially mutagenic events (Li H, Haurigot V, Doyon Y, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature. 475(7355): 217-21, 2011). Levels of blood clotting proteins needed to maintain hemostasis may be more readily achieved by expansion of large populations of cells ex vivo and reintroduction(s) into the patient. Promising work has been done with murine hematopoietic stem cells (HSCs) transduced with lentivirus to express FVIII, including in HA mice with high-titer FVIII inhibitors (Calcedo R, Morizono H, Wang L, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol. 18(9): 1586-8, 2011; Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012). Enthusiasm for these approaches is tempered, however, by recognition that in order to promote engraftment, conditioning agents such as busulfan, which can have serious side effects are required (Chuah M K, Nair N, VandenDriessche T. Recent progress in gene therapy for hemophilia. Hum Gene Ther. 23(6): 557-65, 2012). Furthermore, concerns remain regarding the long-term consequences of lentiviral incorporation into the genomes of hematopoietic stem cells (HSCs).
In addition, there is a critical need to identify ways to avoid FVIII inhibitor development and to abate a FVIII inhibitor response. An arduous (frequent infusions of FVIII) and extremely expensive (˜$1 MM/patient) protocol called Immune Tolerance Induction (ITI) is the only approach proven to eradicate FVIII inhibitors in HA patients, yet fails among 30% of inhibitor patients (Morfin M. et. al. European study on orthopaedic status of haemophilia patients with inhibitors. Haemophiia Sep; 13(5):606-12 2007). Twenty percent of HA patients with the F8I22I develop inhibitors. In addition, HA patients with missense mutations expressed in the C2 domain of FVIII are more prone to inhibitor formation than those with mutations expressed elsewhere in their endogenous FVIII (8.7% C1/C2 domain vs. 3.6% non-C1/C2-domain; p-value: 0.01 sample size 1135 HA patients).